摘要
【目的】构建人回盲肠癌(human ileocecal adenocarcinoma,HCT-8)细胞系的CRISPR/Cas9基因编辑系统,实现对目的基因的高效编辑。【方法】采用慢病毒感染的方式,将慢病毒载体质粒LentiCas9-Blast与包装质粒pSPAX2和pMD2.G共转染至人胚胎肾(human embryonic kidney 293T,HEK293T)细胞系获得高滴度的重组慢病毒液,并感染HCT-8细胞系,以未感染组为阴性对照,用杀稻瘟菌素进行抗性筛选,直至阴性对照组全部死亡;采用有限稀释法对阳性细胞进行单克隆筛选,对筛选得到的单克隆细胞系进行扩大培养,并采用PCR及Western Blot试验验证Cas9基因及其蛋白表达情况,而后对阳性单克隆细胞系进行基因编辑效率及细胞系活性验证。【结果】得到能成功表达Cas9蛋白的6株HCT-8-Cas9单克隆细胞系。其中HCT-8-Cas9_2和HCT-8-Cas9_4细胞系蛋白量表达较高;对所筛选单克隆细胞系进行基因编辑效率检测,pXPR_011慢病毒表达报告基因载体系统检测结果表明,HCT-8-Cas9_4单克隆细胞系Cas9基因编辑效率为48.82%,显著高于其他HCT-8-Cas9单克隆细胞系;单克隆细胞系活性检测结果表明,HCT-8-Cas9_2和HCT-8-Cas9_4单克隆细胞系细胞活性与HCT-8细胞系相比均无显著差异。【结论】HCT-8-Cas9_4细胞系能够稳定表达Cas9蛋白,具有良好的编辑效率,可用于后续靶标基因的高效编辑。
【Objective】This study was conducted to construct a CRISPR/Cas9 gene editing system for human ileocecal adenocarcinoma(HCT-8)cell line to achieve efficient editing of target genes.【Method】With lentiviral infection,LentiCas9-Blast was co-transfected with packaging plasmids pSPAX2 and pMD2.G into human embryonic kidney 293T(HEK293T)cell line to obtain high titer recombinant chronic disease venom.The obtained high titer recombinant lentivirus was used to infect HCT-8 cells.The uninfected group was used as the negative control,and then the resistance was screened with blasticidin until all the negative control groups died.The positive cells were screened by limited dilution method,and the selected monoclonal cell lines were expanded and cultured.The expression of Cas9 gene and protein was verified by PCR and Western Blot,and then the gene editing efficiency and cell lines activity of positive monoclonal cell lines were verified.【Result】We obtained six HCT-8-Cas9 monoclonal cell lines which could successfully express Cas9 protein.Among them,the protein expression of HCT-8-Cas9_2 and HCT-8-Cas9_4 cell lines were higher,and then the gene editing efficiency of the selected monoclonal cell lines was detected.The results of pXPR_011 lentivirus expression reporter gene vector system showed that the gene editing efficiency of HCT-8-Cas9_4 monoclonal cell line was 48.82%,which was significantly higher than that of other HCT-8-Cas9 monoclonal cell lines.The results of monoclonal cell lines activity assay showed that there were no significant differences in cell activity between HCT-8-Cas9_2 and HCT-8-Cas9_4 monoclonal cell lines compared with HCT-8 cells line.【Conclusion】By comparison,HCT-8-Cas9_4 cell line can stably express Cas9 protein and has good editing efficiency,which can be used for efficient editing of subsequent target genes.
作者
李娜
王悦欣
李孝法
王璐阳
梁冠达
李俊强
张龙现
李晓迎
LI Na;WANG Yuexin;LI Xiaofa;WANG Luyang;LIANG Guanda;LI Junqiang;ZHANG Longxian;LI Xiaoying(College of Veterinary Medicine,Ministry of Agriculture and Rural Affairs Key Laboratory for Quality and Safety Control of Poultry Products,Henan Agricultural University,Zhengzhou 450046,China;Henan Sangao Agriculture and Animal Husbandry Co.,Ltd.,Gushi 465200,China)
出处
《河南农业大学学报》
CAS
CSCD
2023年第4期632-638,共7页
Journal of Henan Agricultural University
基金
NSFC-河南联合基金重点项目(U1904203)
河南省高等学校重点科研项目(22A230003)。
